The present invention relates to the field of cell death, and more particularly, to apoptosis. The novel peptides and the compositions comprising said peptides are useful in inhibiting cell death. Therefore, they are potentially useful in treating disorders of inappropriate activation of cell death, such as neurodegenerative disorders, cerebral strokes, myocardial infarctions, etc.
Apoptosis is an intrinsic cell self-destruction or “suicide” program. In response to a triggering stimulus, cells undergo a highly characteristic cascade of events of cell shrinkage, blebbing of cell membranes, chromatin condensation and fragmentation, culminating in cell conversion to clusters of membrane-, bound particles (apoptotic bodies), which are thereafter engulfed by macrophages (Boobis A R, et al. Trends Pharmacol. Sci. 10:275-280, 1989; Bursch W, et al. Trends Pharmacol. Sci. 13:245-151, 1992.).
Normally, apoptosis plays important physiological roles, among others in the development of the central nervous system (Merry D E, et al. Development 10: 301-311, 1994.) However, it is now known that “inappropriate” activation of this death program also plays a critical part in the pathogenesis of numerous disorders, e.g. AIDS, ischemic injuries such as cerebral strokes or myocardial infarctions, and neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease or amyotrophic lateral sclerosis (ALS) (Ziv I, et al. Neuosci Lett. 170: 136-140, 1994; Ziv I, et al. J. Neural. Transm. 49 (supp): 69-75, 1997; Thompson C B. Science 267:1456-1461.). The etiologies of the latter frequent and progressive neurological disorders are unknown. Thus, there are no known therapeutic measures capable of affecting the downhill course of the neuro-degenerative process. However, the substantiation of the role of apoptosis in the neuronal death in these disorders now delineates a novel window for therapeutic interventions, aimed to inhibit the final common biochemical pathway of the apoptotic process, upon which the various triggers of the death program converge.
The Bcl-2 family of proteins is a major system controlling this final common pathway. This growing family of proteins includes death-inhibitory members (Bcl-2, Bcl-xL, Bcl-w, Ced-9, Mcl-1, Al) as well as death inducers (Bax, Bak, Bcl-xS, Bad, Bik, Bid, Hrk) (Kroemer G. Nat. Med. 3:614-620, 1997; Reed J C. Nature 387:773-776, 1997.) This protein system has been shown to be a powerful regulator of cell death. Bcl-2 can protect cells from a wide array of insults, and can inhibit both apoptotic and necrotic modes of cell death (Shimizu S. Nature 374:811-813, 1995; Ziv I, et al. Apoptosis 2:149-155, 1997). On the other hand, transgenic Bcl-xL-knock-out mice manifest extensive apoptosis of neuronal tissues (Motoyama N, et al., Science 267:1506-1510), whereas neurons of Bax knock-out mice manifest resistance to apoptosis (Deckwerth T L, et al., Neuron 17:401-411, 1996). Clinical relevance of this protein system is reflected, among others, in reports of Bax upregulation following cerebral ischemia (McGibbon G A, et al. Brain Res. 750:223-234, 1997) and also in Alzheimer's disease brains and ALS spinal cord motor neurons (Su J H, et al. J. Neuropathol. Exp. Neuro. 56:86-93, 1997; Mu X, et al. Ann Neurol 40:379-386, 1996).
The members of the Bcl-2 family of proteins are strategically localized in the outer mitochondrial membrane, endoplasmic reticulum, nuclear envelope, and the cytosol (Kromer G. Nat Med 3:614-620, 1997; Reed J C Nature 387:773-776, 1997). Bcl-xL has a predominantly mitochondrial localization. Notably, Bcl-xL manifests high levels of expression in the central nervous system (Mizuguchi M, et al. Brain Res 712:281-286, 1996). Bax, a major death inducer, is predominantly cytosolic, but manifests redistribution to the mitochondria upon induction of apoptosis (Wolter K G, et al., J Cell Biol 139:1281-1292, 1997.
Amino acid sequence analysis of the Bcl-2 family yielded a focus on several regions within the proteins (Yin X M, et al. Nature 369:321-323, 1994; Sedlak T W, et al., Proc. Natl. Acad. Sci. USA, 92:7834-7838, 1995; Cheng E H, et al., Nature 379:554-556, 1996; Chittenden, T et al., EMBO J. 14:5589-5596, 1995; Hunter J. at al. J. Biol. Chem. 271:8521-8524, 1996; Wang K, et al., Genes Dev. 10:2859-1869, 1996). These regions are:    1. A hydrophobic C-terminal region, which serves for membrane anchoring.    2. BH1 and BH2: Regions which are important for formation of a hydrophobic binding cleft, where protein—protein interactions take place.    3. BH3: The C-terminal half of the amphipathic Bcl-xL second helix, serves as part of the hydrophobic binding cleft. The homologous region in the death-inducing family members, serves as a ligand region, and is important for their protein—protein interactions with other proteins within the family.    4. A flexible, cytosol-exposed PEST-like region in Bcl-2 and Bcl-xL, which serves as a regulator region. It includes serine phosphorylation sites.    5. BH4: an N-terminal region, which serves to stabilize the three dimensional protein structure, as well as a critical docking region for several proteins, e.g., Raf-1, Bag-1 and Ced-4.
The mode of action of the Bcl-2 family proteins in the regulation of cell survival is largely unknown, though two major; functions have been revealed:    1. Adaptor/Docking proteins: Bcl-2 and B1-xL, by virtue of their membrane attachment with cystolic orientation, have been shown to act as important adaptor or docking proteins, pulling-out proteins from the cytosol, thus inactivating them or orienting them to interact with other membrane-bound proteins. Among these are the protein kinase Raf-1, calci-neurin, R-Ras, H-Ras, the prion protein Pr-1, Bag-1, the p53-binding protein p53-BP2 and others (Kroemer G. Nat Med 3:614-620, 1997; Reed J C. Nature 387:773-776, 1997). Another important protein shown to be docked to Bcl-2 is the Apaf-1 protein. This death-inducing protein, homologous to the nematode Caenorhabditis-elegans protein CED-4, acts in mediating linkage between the Bcl-2 system and the downstream cysteine proteases (caspases), which perform the execution phase of the death program. (Zou H et al., Cell 90:405-413, 1997; Yuan J., et al., Development 116:309-320, 1992; Chinnaiyan A M, et al., Science 275:1122-1126, 1997)    2. Formation of transmembrane pores and/or ionic channels, as suggested by the similarity of the crystal structure of Bcl-xL and the structure of pore forming bacterial toxins, e.g. colicins and diphtheria toxin (Muchmore, et al., Nature 381:335-344;1996). Bcl-xL, Bcl-2 and Bax have all been shown to be capable of transmembrane ionic channel formation, the two formers only in acidic pH, whereas the latter also in physiological pH. (Minn A J, et al. Nature 385:353-357, 1997; Schendel S L, et al. Proc. Natl. Acad. Sci. USA 94:5113-5118, 1997; Antonsson B et al., Science 277:370-372, 1997; Schlesinger P H, et al., Proc. Natl. Acad. Sci. USA 94:11357-11362, 1997).
These structure-function considerations and the localization of the Bcl-2 and Bcl-xL to the outer mitochondrial membrane, are in accordance with the emerging importance of the mitochondrial level in the apoptotic cascade (Zamzami N, et al. J. Exp Med 183:1533-1544, 1996). Disruption of the mitochondrial transmembrane potential has been shown to be an early event in apoptosis (Zamzami N, et al., J. Exp Med 181:1661-1672, 1995). Evidently, this derangement involves the opening of so-called mitochondrial permeability transition pores (PTP) (Zamzami N, et al., J Exp Med 182:367-377,1995). These are megachannels, which can be opened in response to numerous noxious stimuli and lead to redistribution of molecules of <1,500 daltons, thus disrupting mitochondrial membrane potential and associated mitochondrial functions (Zoratti M, et al., Biochim Biophys Acta 1241:139-176, 1995). Chemical inducers and inhibitors of the PTP have been shown to induce or inhibit apoptosis, respectively (Marchetti P, et al., Apoptosis 1:119-215, 1996; Zamzami N, et al., FEBS Letters 384:53-57, 1996). Moreover, during the apoptotic process, mitochondria have been shown to liberate at least three mediators of apoptosis: ceramide, cyctochrome c, and AIF; (apoptosis-inducing factor) a 50 kDa protein with caspase-like activity (Marchetti P, et al., Apoptosis 1:119-125, 1996). Interestingly, all these mitochondrial-dependent steps in the apoptotic process have been effectively blocked by Bcl-2 (Zamzami N, et al., J Exp Med 182:367-377, 1995; Kluck R M, et al., Science 275:1132-1136, 1997; Susin S, et al., J Exp Med 184:1331-1342, 1996).
Specific importance is currently attached to the release from the mitochondria of cytochrome C, which acts to activate the downstream apoptotic cascade, and can be liberated and exert its pro-apoptotic activity even in the absence of mitochondrial potential collapse (Bossy-Wetzel E, et al. EMBO J, 17:37-49, 1998; Li F, et al J Biol Chem, 272:30299-30305,1997).
The Bcl-2 system is therefore a powerful system in determining cell fate. For purposes of the development of novel diagnostics and therapeutics for modulation of cell death, it has therefore been desirable to identify specific regions or domains within these proteins which are, by themselves, capable of fulfilling at least part of the functions of the whole proteins. For example, the identification of agents capable of mimicking the apoptosis-inhibitory effects of Bcl-2 and Bcl-xL may be useful for the treatment of medical disorders associated with inappropriate activation of the death program, for example, neurodegenerative disorders, ischemic injury (cerebral stroke, myocardial infarction), AIDS, myelodysplastic syndromes, traumatic or toxic injuries.